Shortle D R, Margolskee R F, Nathans D
Proc Natl Acad Sci U S A. 1979 Dec;76(12):6128-31. doi: 10.1073/pnas.76.12.6128.
The circular genome of simian virus 40 is a model mammalian replicon, containing a unique origin of replication (ori) and coding for a protein (SV40 T antigen) known to be involved in initiation of viral DNA replication and to bind in vitro to the origin region. Mutations within the ori sequence lead to defective viral DNA replication and the formation of small viral plaques after infection of a cell monolayer. Second-site revertants (pseudorevertants) of ori mutants were isolated by random local mutagenesis of mutant DNA followed by transfection of cultured cells and the selection of large plaques. In each case, reversion of the plaque phenotype was associated with an increased rate of viral DNA replication. The second-site mutations that suppressed the replication defects were localized by in vitro recombination or marker rescue experiments to the gene for T antigen. Their map positions differ from those of previously described T antigen mutants, possibly reflecting a specific ori-binding domain of T antigen. From these results we infer that T antigen interacts with the ori signal during virus development as it does in vitro and that this interaction regulates the rate of viral DNA replication.
猴病毒40的环状基因组是一种典型的哺乳动物复制子，含有一个独特的复制起点（ori），并编码一种蛋白质（SV40 T抗原），已知该蛋白质参与病毒DNA复制的起始过程，并在体外与起点区域结合。ori序列内的突变会导致病毒DNA复制缺陷，并在感染细胞单层后形成小的病毒蚀斑。通过对突变DNA进行随机局部诱变，然后转染培养细胞并筛选大蚀斑，分离出ori突变体的第二位点回复突变体（假回复突变体）。在每种情况下，蚀斑表型的回复都与病毒DNA复制速率的增加有关。通过体外重组或标记拯救实验，将抑制复制缺陷的第二位点突变定位到T抗原基因。它们的图谱位置与先前描述的T抗原突变体不同，这可能反映了T抗原的一个特定ori结合结构域。从这些结果我们推断，T抗原在病毒发育过程中与ori信号相互作用，就像它在体外那样，并且这种相互作用调节病毒DNA复制的速率。
